Site-directed mutagenesis. See

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

RCs from Rb.sphaeroides have been altered by site-directed mutagenesis (see for example ref. 18). Crystals have been grown of RCs that have been altered at L212 (see ref. 18) and M210 (in collaboration with C. Schenck). These crystals have the same space group as the native RCs, comparable cell parameters, and diffract to approximately 3.5 A. Collection of diffraction data from these crystals is in progress. 

Virus expressing JHE modified by site-directed mutagenesis (see text). 

Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

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